Differences in the Mechanism of In Vitro Immune Hemolysis Related to Antibody Specificity*

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Abstract
This study was undertaken to determine whether complement-dependent antibody injury to human erythrocytes results in hemolysis by a colloid-osmotic mechanism, as well as the size of the antibody-complement induced membrane defect. Human erythrocytes were incubated with rabbit anti-human red cell serum or complement-fixing human anti-A or immune anti-A from rabbits and fresh autologous human serum. The amount of hemolysis produced was compared to the percent of erythrocyte potassium lost into the medium. The mechanism of hemolysis was investigated by addition of osmotically active macromolecules, in extracellular concentrations sufficient to balance the osmotic pressure of intracellular hemoglobin, permitting potassium loss while preventing hemoglobin escape from the cells if the membrane defect was smaller than the protective macromolecule. Bovine serum albumin (Effective Diffusion Radius - RES 35.5 A), Dextran 40(RES >32.5 A), Dextran 20 (RES 32 A) and Dextran 10 (RES 22.2 A) were used. Results: (1) Rabbit antihuman red cell serum and human complement (C'') produce functional holes in human erythrocyte membranes which are 32 A in effective radius. Destruction of these injured cells in vitro ensues by colloid-osmotic lysis. (2) In contrast anti-human A of rabbit and human origin with human complement produces holes greater than 32.5 A in radius, permitting direct escape of hemoglobin. (3) The size of the membrane defect is not related to either molecular size of the antibody molecules, or the species of animal producing the antibody. (4) It is suggested that specificity of the antigenic sites involved determines the size of the membrane defect produced. In turn, the mechanism of the complement dependent hemolysis is determined by the size of the induced membrane defects.